Abstract
Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E.coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.
Highlights
Silks are a general term for widely different protein polymers spun by a range of different arthropods with a range of functions [1]
The B. mori cocoonase (BmCoc) cDNA was cloned into either (1) a pETHis-1a vector, providing a tobacco etch virus (TEV) protease cleavable N-terminal poly histidine tag (His-tag) or (2) a pET-NHis vector providing a N-terminal His-tag with an enterokinase cleavable linker
GAM-BmCoc serves as a proxy of the inacti vated zymogen and was designed to enable structural studies since it is expected to have a lower likelihood of self-cleavage
Summary
Silks are a general term for widely different protein polymers spun by a range of different arthropods with a range of functions [1]. With a global production of more than 150,000 tons a year, from 1 million tons of fresh cocoons, silk is one of the most valuable natural fibers in the world [2], with an annual production value of more than 3 billion dollars in 2019 (fao.org/faostat/, FAO 2020). One crucial step in the process of turning the cocoon into fabric is the removal of the protein glue, sericin that holds the fibroin fibers together in the cocoon, this process is called degumming [3]. The silk moth uses the protease cocoonase to degrade sericin to soften the cocoon. This is essential for the fully developed moth to be able to escape the cocoon [4]. Cocoonase has a high potential for improving the effectivity of degumming, while at the same time reducing the envi ronmental impact of the silk industry
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